The emergence of highly pathogenic avian influenza viruses (AIVs) poses significant threats to global public health and the poultry economy. Rapid detection methods targeting the hemagglutinin (HA) protein have become increasingly unreliable due to ongoing antigenic drift and genetic mutations. In this study, we screened two monoclonal antibodies (mAbs) against the highly conserved neuraminidase (NA) protein, which were characterized by high affinity and specificity for the N9 subtype. Using the paired mAbs 12A2 and 12F12, an NA-targeting lateral flow immunoassay (NA-LFIA) was developed for the rapid detection of N9 subtype AIVs. The NA-LFIA demonstrated exceptional specificity, exhibiting no cross-reactivity with the N1, N2, or N6 subtypes. The limits of detection (LODs) were established at 103.3 TCID50/0.1 mL for the virus and 98 ng/mL for the recombinant NA protein. The test strips exhibited excellent reproductivity for detecting the NA antigen, with intra-assay and inter-assay variations of 5.32% and 6.05%, respectively. Stability testing confirmed a six-month shelf life without any loss of sensitivity. These finds indicate that the NA-LFIA is a robust, rapid point-of-care testing (POCT) tool for the early detection of N9 subtype AIV infection.

Senecavirus A (SVA) is an emerging swine virus with global prevalence that causes vesicular disease (VD), clinically similar to foot-and-mouth disease (FMD), posing a significant concern for the swine industry. The capsid protein VP2 is a structural protein of SVA, playing a critical role in mediating viral entry into host cells and inducing the production of neutralizing antibodies. In this study, the SVA VP2 protein was expressed using the Bac-to-Bac baculovirus expression system. Six monoclonal antibodies (mAbs) targeting SVA VP2 protein were then produced by immunizing mice with the recombinant VP2 protein, named as 1A1F6, 3D5F9, 3E2C3, 5A6F5, 5F12D10 and 7H10C3, respectively. Among these, mAbs 1A1F6 and 7H10C3 exhibited neutralizing activity against SVA in vitro with IC50 values of 0.64 μg/mL and 1.21 μg/mL, respectively. Finally, a linear B-cell neutralizing epitope of 151SLQELN156 on the SVA VP2 protein was identified by determining the reactivity of the neutralizing mAbs with the truncated VP2 protein followed by peptide scanning. Peptide mutation analysis showed that the residues Ser151, Leu152, Leu155, and Asn156 within the epitope were essential for antibody binding. Multiple sequence alignment indicated that this epitope is highly conserved across various SVA strains. These findings provide a foundation for further studies on SVA and offer valuable support for the design of SVA vaccines.

African swine fever (ASF) is a highly contagious viral disease threatening global swine industries. Rapid and accurate detection of ASF virus (ASFV) antibodies is crucial for disease surveillance and control. The gold lateral flow immunoassay (GLFIA) is cost-effective and has been successfully applied in rapid on-site detection of ASFV. However, its sensitivity is relatively low. To enhance the detection sensitivity and accuracy while retaining convenience, we developed a chemiluminescent lateral flow immunoassay (CLFIA) for detecting ASFV antibodies based on the p72 trimer protein, which can immediately read the chemiluminescent signal through the camera of a smartphone. Compared with GLFIA and commercial enzyme-linked immunosorbent assay (ELISA), its sensitivity was improved by at least two orders of magnitude and nine orders of magnitude, respectively. Additionally, CLFIA shows no cross-reaction with antibodies from common swine disease viruses, and the detection results of 65 clinical samples have a 93.8% coincidence rate with those of commercial ELISA kits. This research successfully addressed the issue that traditional chemiluminescent detection relies on specialized instruments, providing a new technical approach for the highly sensitive and rapid detection of ASFV, and effectively promoting the development and application of CLFIA technology.

BACKGROUND:Pseudorabies (PR) is a highly contagious disease, and it causes significant economic losses to the global swine industry. Vaccination plays an important role in the prevention and control of pseudorabies virus (PRV). To evaluate vaccine efficacy, there is a need for a quick and straight forward method to monitor PRV-induced antibody levels in practice. RESULTS:A time-resolved fluorescence immunochromatographic (TRFIC) strip was developed for the serological detection of PRV gB antibodies in swine. Following systematic analysis and evaluation, the assay demonstrated a high degree of correlation with established reference method. The positive and negative coincidence rates between the TRFIC strip and ELISA were 96.8% and 94.2%, respectively. Furthermore, comprehensive analytical and comparative assessments revealed that the TRFIC strip exhibited no cross-reactivity with antibodies against other porcine pathogens. CONCLUSION:Given its high specificity, sensitivity, and convenience, the TRFIC strip is suitable for on-site detection of PRV gB antibodies and can serve as a valuable tool for monitoring PRV immune status in animal populations.

Aflatoxin B1 (AFB1), a highly toxic and carcinogenic mycotoxin, poses significant public health risks due to its widespread contamination of staple food crops such as peanuts and maize. Although conventional lateral flow immunoassays (LFIAs) are widely employed for rapid on-site screening, their limited sensitivity frequently compromises accurate quantification at trace levels. To improve the analytical performance of LFIAs, we developed a novel time-resolved fluorescence-based lateral flow immunoassay (TRFN-LFIA) by integrating reverse artificial antigen labeling with time-resolved fluorescence signal amplification. This method enhances detection sensitivity and enables rapid, ultra-sensitive, visible, and quantitative determination of AFB1 in peanut and maize samples. Under optimized conditions, the TRFN-LFIA achieved a visible limit of detection (vLOD) of 0.30 ng/mL (2.22 µg/kg), a quantitative limit of detection (qLOD) of 0.04 ng/mL (0.30 μg/kg), and a half-maximal inhibitory concentration (IC50) of 0.09 ng/mL. Recoveries from spiked peanut and maize samples ranged from 81.33% to 117.86%, with coefficients of variation (CVs) below 13.04%. Analysis of 21 real samples (13 maize and 8 peanut samples) yielded results highly consistent with those obtained by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Moreover, the method demonstrates significant advantages in terms of detection speed, cost-effectiveness, and operational convenience. Therefore, the results established the TRFN-LFIA method as a reliable and practical tool for on-site rapid detection of AFB1 in contaminated food matrices, providing both a rapid and accurate approach for trace-level quantification and a novel strategy for enhancing the sensitivity of lateral flow immunoassays.

Zearalenone (ZEN), a stable mycotoxin with estrogenic activity produced by various Fusarium species, poses a serious food safety risk. To facilitate the rapid, sensitive, on-site detection of ZEN in maize and ensure consumer dietary safety, a colloidal gold immunochromatographic assay (CG-ICA) based on a monoclonal antibody was established. ZEN was converted via oxime derivatization into hapten ZAN-O, which was conjugated to a carrier protein to prepare an immunogen for producing a highly specific and sensitive monoclonal antibody. Then, the antibody was conjugated into colloidal gold nanoparticles (AuNPs) and used as capture bioprobes of the CG-ICA test strip. The highly sensitive and specific detection platform was established through systematic optimization of pH value, coating antigen concentration, antibody-labeling dosage, incubation time, and strip assembly conditions. Under optimized conditions, the strip exhibited a detection limit of 11.79 pg/mL and an IC50 of 99.06 pg/mL, with a linear detection range of 13.40-732.48 pg/mL. In addition, the anti-interference capability assay demonstrated that the developed test strip possessed excellent specificity. In spiked maize samples, the CG-ICA test strip demonstrated recoveries ranging from 85.36% to 98.86%, with relative standard deviations (RSDs) below 10%. Thus, the CG-ICA strip provides a rapid, sensitive, and robust on-site tool for ZEN screening in maize, and can be adapted to other hazards by simply switching the antibody.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the seasonal influenza virus are spreading among humans concurrently, especially with the ongoing replacement of mutant strains. It is challenging to differentiate between symptoms for therapy due to the comparable symptoms following infection with the SARS-CoV-2 variants and influenza viruses. Meanwhile, in order to achieve rapid point-of-care testing (POCT) to manage the spread of the disease, we developed a dual lateral flow strip based on colloidal gold- labeled monoclonal antibodies that can perform differential detection of SARS-CoV-2 variants and influenza A viruses (IAV) in this study. High-affinity monoclonal antibodies (mAbs) targeting SARS-CoV-2 and IAV were prepared to capture antigens and labeled with colloidal gold nanoparticles (AuNPs). Based on high-affinity mAbs, two targets were immobilized on one nitrocellulose (NC) membrane to establish the lateral flow strip (LFS) for differential diagnosis of SARS-CoV-2 and IAV. With no reactivity to other viruses, this LFS is extremely specific and can only identify SARS-CoV-2 and IAV. The LFS showed a limit of detection (LOD) of 4.88 ng/mL for the Omicron BA.2 RBD protein and 2.44 ng/mL for the nucleoprotein (NP) protein of H1N1. When analyzing 16 SARS-CoV-2 positive clinical samples, eight IAV positive clinical samples, and six negative samples that had already been pre-confirmed by commercial kits, its clinical application is effectively and accurately proven. These results demonstrated that the LFS integrated with AuNPs has great potential to facilitate quick, easy, and reliable POCT diagnosis for promoting the control of infectious diseases.

To establish a highly sensitive chemiluminescent enzyme-linked immunosorbent assay (ci-ELISA) method for the determination of florfenicol (FF) in egg and chicken meat. Based on the obtained monoclonal antibodies against florfenicol, a chessboard test was employed to determine the optimal working concentration of artificial antigen and antibody, and then established a ci-ELISA method, and characterized its performance. The results showed that the ci-ELISA method had a detection limit of 5.433 pg/mL, an IC50 of 30.893 pg/mL, and a detection range between 5.433 and 193.577 pg/mL. The coefficient of variation was below 10

6-benzylaminopurine (6-BA) is a widely used plant growth promoter. However, it can lead to precocious puberty and cause acute poisoning and even cancer of consumer. Thus, it is necessary to establish rapid and sensitive method for the detection of 6-BA. Electrochemical immunosensor is a new kind of biosensor, which combines the high selectivity of antibody with the high sensitivity of electrochemical sensor. In this study, an electrochemical immunosensor was developed for the detection of 6-BA in bean sprouts. The carbon material of Zn/Ni-ZIF-8-800 derived from bimetal-organic framework was synthesized by solvothermal method. Then, Zn/Ni-ZIF-8-800 was ultrasonically mixed with the highly conductive graphene, and the gold nanoparticles (AuNPs) were reduced by constant potential deposition (AuNPs/Zn/Ni-ZIF-8-800 @graphene). Finally, the antibody of 6-BA was combined with the prepared material to make an electrochemical immunosensor. The prepared materials were charac-terized by XRD, FTIR, SEM, TEM and XPS analyses. In addition, the electron transfer abilities of the modified sensors were investigated by CV and EIS. The established immunosensor presented a wide linear range (0.08-50 ng/mL), low detection limitation (0.24 ng/mL) and satisfactory recovery rates towards 6-BA (90.9-105.0 %). In brief, an electrochemical immunosensor with high sensitivity and selectivity was developed, which could be used to detect 6-BA in bean sprouts effectively.

In this study, a sensitive immunochromatographic assay (ICA) was developed for simultaneously detecting azaperone (AZN) and its metabolite azaperol (AZL) based on the high-affinity monoclonal antibody (mAb). Herein, the hapten AZL-SA was synthesized by succinic anhydride method, and then the conjugates AZL-SA-OVA and AZL-SA-KLH were prepared by EDC/NHS method. Subsequently, mAb was produced for targets monitoring through two detection modes. In direct ICA (using gold nanoparticles labeled specific antibodies), the visual LOD of AZN was 80 ng/g. For indirect ICA (using gold nanoparticles labeled anti-species antibodies), the visual and instrumental LODs of AZN were 8 and 0.14 ng/g, and AZL were 8 and 0.12 ng/g, respectively. The results indicated that the visual detection limit (LOD) of indirect format was tenfold lower than that of direct format. The established analytical method obtains the results within 15 min and provides a sensitive and simple tool for on-site detection of AZN and AZL.

An ultrasensitive and broad-specific monoclonal antibody recognising cyproheptadine hydrochloride and six phenothiazines was produced. The 50% inhibition concentration against cyproheptadine hydrochloride was 0.036 ng/mL, and the cross-reactivities for six phenothiazines were from 6.33% to 63.16%. Based on the developed monoclonal antibody, an immunochromatographic strip was established, with the visual detection limits (cut-off values) of seven drugs ranging from 5 to 100 ng/g in feedstuffs. With the strip reader, the 50% inhibition concentration of the developed immunochromatographic strip for seven drugs ranged from 0.570 to 7.750 ng/g. The intra-assay recoveries were from 79.8% to 103.4% with the highest coefficient of variation of 11.3%. The inter-assay recoveries were from 79.0% to 96.6% with the highest coefficient of variation of 12.7%. In summary, the proposed immunochromatographic strip was considered suitable for simultaneously monitoring cyproheptadine hydrochloride and phenothiazines in feedstuffs.

African Swine Fever (ASF) is an acute and highly lethal disease in pigs caused by African Swine Fever Virus (ASFV). Viral proteins have been commonly used as antigenic targets for the development of ASF diagnostic methods. However, the prokaryotic expression of viral proteins has deficiencies such as instability, insolubility, and high cost in eukaryotic situations. This study screened and verified ASFV-encoded p72, p54, and p30 protein antigenic epitopes. Subsequently, a novel antigenic epitope-associated recombinant protein was designed based on an ideal structural protein and expressed in Escherichia coli (E. coli). Western blot analysis indicated that the recombinant protein could specifically react with the monoclonal antibody (mAb) of p72 and polyclonal anti-bodies of p54 and p30, respectively. Next, an ASF indirect ELISA (iELISA) method was established based on the recombinant protein, which has no specific reaction with sera of other important pig viral diseases. Meanwhile, it shows a sensitivity to detecting dilutions of ASF-positive reference serum up to 1:6400. The clinical sample detection results showed a high coincidence rate of 98 % with a commercial competition ELISA kit. In conclusion, we established a novel specific, and sensitive ASF serologic detection method that opens new avenues for ASF serodiagnostic method development.

Porcine circovirus type 2 (PCV2) Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. Although many methods can be used to identify PCV2 or PCV2 Cap protein in vaccine research, they usually require high workload and time.

为了研制灵敏、准确、特异及稳定的非洲猪瘟抗体快速检测试纸产品,以非洲猪瘟病毒(ASFV)p72蛋白为检测抗原,以金黄色葡萄球菌A蛋白(SPA)为拦截线,对比重组蛋白p72的不同状态对试纸检测性能影响,优化标记条件、喷膜浓度、样品垫缓冲体系及成分、显色时间等因素,确定ASFV抗体检测试纸产品化的具体参数,并对试纸的特异性、均一性、准确性及稳定性进行鉴定.结果显示,试纸的检测敏感性为1∶51 200,优于商业化ASFV检测试剂盒,与其他猪源病毒阳性血清无交叉反应,变异系数小于10%,在临床样本检测中未见假阴性及假阳性结果,具有良好的敏感性、特异性、均一性及准确性;经加速稳定试验验证,可以室温保存1a以上;与进口商品化试剂盒的总符合率为91.6%.综上,成功研制了ASFV抗体检测试纸,且试纸的检测性能良好,可以用于ASFV抗体的快速筛查.

[目的]建立一种基于量子点(quantum dots,QDs)技术的猪伪狂犬病病毒(Pseudorabies virus,PRV)gB抗体免疫层析试纸,为PRV疫苗免疫效果评估提供一种快速、简便的检测技术.[方法]选用昆虫细胞表达系统表达的gB蛋白,采用羧基修饰的水溶性QDs,在偶联剂1-乙基-3[3-二甲基氨基丙基]碳二亚胺盐酸盐(EDC)的作用下制备gB-QDs荧光标记物.将金黄色葡萄球菌蛋白A(Staphylococcus aureus protein A,SPA)和抗gB蛋白的单克隆抗体分别固定在硝酸纤维素膜上作为检测线和质控线,将样品垫、标记垫、吸水垫、支撑底板按生产工艺组装成基于QDs的荧光免疫层析试纸.检测该试纸的特异性、敏感性及与商品化ELISA试剂盒的符合率.[结果]敏感性试验结果显示,该试纸的敏感性为1∶6 400.特异性试验结果显示,该试纸与猪瘟病毒(CSFV)、口蹄疫病毒(FMDV)、猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)阳性血清均无交叉反应,特异性为100％.通过检测田间猪血清样品,对比商品化ELISA检测试剂盒,结果显示,113份田间猪血清样品中,两种方法检测结果不一致的血清有11份,结果一致的血清有102份,该试纸与商品化ELISA试剂盒的符合率为90.3％.[结论]本研究制备的试纸检测线紫外灯下显色清晰可见,辨识度高,具有较高的特异性和敏感性,可用于PRV gB抗体的检测.

花生是主要的油料作物之一,也是消费者宝贵的蛋白质来源,但却会在过敏个体中引起过敏反应,甚至引起死亡.目前,过敏反应尚缺乏准确的治疗方案,避免食用致敏食品是保障过敏患者食品安全的主要途径.因此,加强对食品中花生致敏蛋白的监测对于食品致敏性风险评估与监管尤为重要.该文分别从蛋白质和DNA水平对花生致敏蛋白检测方法进行综述,并对其发展趋势进行展望,以期为花生致敏蛋白检测方法的进一步研究与应用提供参考.

为了建立更灵敏的玉米赤霉烯酮(zearalenone,ZEN)快速检测方法,以量子点(quantum dots,QDs)为标记材料,利用活泼酯法标记ZEN单克隆抗体制备荧光免疫探针,以ZEN-BSA为检测线,成功制备了 ZEN荧光免疫层析试纸.结果表明,该试纸回归方程为y=-0.532 5x+0.937 2,IC50为6.6ng/mL,检测限为1.2ng/mL;与玉米赤霉酮(ZAN)交叉反应率为88.2％,与其他结构类似物交叉反应率均小于10％,与常见真菌毒素黄曲霉毒素B1(aflatoxin B1,AFB1)、脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)、T-2均无交叉反应;玉米样品加标回收率为86.4％-108.5％,变异系数小于15％;通过高效液相色谱(HPLC)法确证,本研究建立的荧光免疫层析试纸条可用于玉米中ZEN的快速筛查.

African swine fever virus (ASFV) causes a highly contagious and often lethal swine viral disease, and leads to tremendous economic losses to the swine industry. Unfortunately, there are no vaccines and effective antiviral agents available to prevent and control ASFV outbreaks. Therefore, it is necessary to develop simple and rapid strategies to monitor ASFV-infected pigs to restrain its spread. In the current study, ASFV capsid protein p72 was expressed along with its chaperone pB602L to form trimers in human embryonic kidney 293 (HEK293) cells. The p72 trimers were subsequently labeled with colloidal gold to develop a immunochromatographic strip. The strip showed high specificity to ASFV-positive serum and no cross-reactivity to other swine virus positive sera. Importantly, the strip showed a higher sensitivity of detecting ASFV antibodies in both positive standard serum and clinical serum samples than a commercial enzyme-linked immunosorbent assay (ELISA) kit. Taken together, these results demonstrate the strip as a reliable diagnostic tool against ASFV infection, which will be appropriate for application in prevention and control of ASFV. • ASFV p72 trimers were successfully generated. • A colloidal gold strip was developed based on ASFV p72 trimers. • The strip is appropriate for detecting ASFV antibodies in the field.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can be transmitted from human to companion animals. The national wide serological surveillance against SARS-CoV-2 was conducted among pet animals, mainly in cats and dogs, 1 year after the first outbreak of COVID-19 in China. All sera were tested for SARS-CoV-2 IgG antibodies using an indirect enzyme linked immunosorbent assay (ELISA) based on the receptor binding domain (RBD) of spike protein. This late survey takes advantage of the short duration of the serological response in these animals to track recent episode of transmission. A total of 20,592 blood samples were obtained from 25 provinces across 7 geographical regions. The overall seroprevalence of SARS-CoV-2 infections in cats was 0.015% (2/13397; 95% confidence intervals (CI): 0.0, 0.1). The virus infections in cats were only detected in Central (Hubei, 0.375%) and Eastern China (Zhejiang, 0.087%) with a seroprevalence estimated at 0.090 and 0.020%, respectively. In dogs, the seroprevalence of SARS-CoV-2 infections was 0.014% (1/7159; 95% CI: 0.0, 0.1) in the entire nation, seropositive samples were limited to Beijing (0.070%) of Northern China with a prevalence of 0.054%. No seropositive cases were discovered in other geographic regions, nor in other companion animals analyzed in this study. These data reveal the circulation of SARS-CoV-2 in companion animals, although transmission of the virus to domestic cats and dogs is low in China, continuous monitoring is helpful for the better understand of the virus transmission status and the effect on animals.